Genetic diversity among natural populations of Keteleeria evelyniana Mast. in Tay Nguyen of Viet Nam using SSR markers - Tran Thi Lieu

Vietnam Journal of Science and Technology 56 (3) (2018) 275-285 DOI: 10.15625/2525-2518/56/3/9820 GENETIC DIVERSITY AMONG NATURAL POPULATIONS OF Keteleeria evelyniana Mast. IN TAY NGUYEN OF VIET NAM USING SSR MARKERS Tran Thi Lieu * , Vu Thi Thu Hien, Dinh Thi Phong Vietnam National Museum of Nature, VAST, 18 Hoang Quoc Viet, Cau Giay, Ha Noi * Email: tranthilieu@gmail.com Received: 16 May 2017; Accepted for publication: 16 March 2018 Abstract. Keteleeria evelyniana Mast. is a big softwood species with high economic values. Therefore, the number of individuals are rapidly decreasing due to rampant exploitation as well as its habitat loss and recently, the species is considered vulnerable in Viet Nam. In this study, we assessed the genetic variation among seventy K. evelyniana samples of three natural populations in Lam Dong, Dak Lak and Kon Tum using 16 microsatellite markers. The results showed that thirteen markers were polymorphic. A total 39 DNA fragments were amplified, among them, thirty – five were polymorphic (accounting for 89.74 %). Among studied populations, the level of genetic diversity at Lam Dong (Na = 2.063; Ne = 1.730; Ap = 0.375; I = 0.558; Ho = 0.459 and He = 0.367) was the highest. Analysis of molecular variance (ANOVA) showed that the total level of molecular changes between populations was 34.65 % and between individuals in the same population was 65.35 %. Private alleles (Ap) and inbreeding values (Fis) of K. evelyniana species were founded of all three populations in Lam Dong, Dak Lak and Kon Tum (0.375 and - 0.234; 0.188 and - 0.065; 0.063 and - 0.047, respectively). The gene flow (Nm) also occurred among the K. evelyniana populations with the average of Nm = 5.423. A dendrogram (UPGMA) constructed based on the similarity matrix of 70 K. evelyniana samples divided into two main groups with their genetic similarity coefficient ranged from 76.5 % (Ke26 and Ke44) to 99 % (Ke23 and Ke25). The obtained results indicated the importance of conserving the genetic resources of K. evelyniana species in Tay Nguyen. Keywords: Keteleeria evelyniana, population genetic diversity, species conservation, SSR, Tay Nguyen. Classification numbers: 1.3.2; 3.1.2. 1. INTRODUCTION Tay Nguyen is the central highlands of Viet Nam and covers two of six phytogeographic sub-regions of Viet Nam [1], including the sub-region South Truong Son and South Indochina in Kon Tum, Gia Lai, Dak Lak, Dak Nong and Lam Dong provinces. According to Nguyen Tien Hiep et al. [2], among the 34 known coniferous species of Viet Nam, there were 15 species of high economic and scientific value in Tay Nguyen, including Keteleeria evelyniana of the Tran Thi Lieu, Vu Thi Thu Hien, Dinh Thi Phong 276 Keteleeria genus in the Pinaceae family. It is an evergreen tree that can grow up to 25 - 30 (or 40) metres high. Its trunk is straight and can expand up to two metres in diameters. The mature trees have broad crown, grey bark and longitudinally fissured. The species is distributed mainly at altitudes ranging from 500 to 2000 metres above sea-level, in mixed conifer/broad-leaved forest, scattering from the North West Viet Nam to Tay Nguyen. In Tay Nguyen, K. evelyniana is currently found only in Lam Dong (at Suoi Vang, Da Chais and Hiep An regions of Lac Duong and Duc Trong districts, respectively), Dak Lak (Hoa Son, Krong Bong district) and Kon Tum (Dak Glei district). Beside Viet Nam, the species can also be found in China and Laos. However, due to over-exploitation and habitat loss, the species is becoming endangered. Currently, the number of mature trees in the wild is small, although the species had been described in the past and was harvested for timber, oil and resin. Based on data collection of surveys and ranking standards in the IUCN Red List, this species can be considered as vulnerable - VU A4acd, B1 + 2b (ii , iii, v), C [3]. Therefore, a study on genetic diversity of K. evelyniana should urgenlly be conducted in order to provide information for an effective conservation strategy in Tay Nguyen. Genetic diversity analysis can be done based on morphological, biochemical, and molecular types of information. However, molecular markers have advantages over other kinds, where they show genetic differences on a more detailed level without interferences from environmental factors. Application of molecular markers in genetic diversity studies have been began from the 1980s and many techniques had developed. Among the molecular markers, SSR (Simple Sequence Repeats) are most popular because it is a codominant marker with high polymorphism and specificity. Therefore, it was considered highly effective in the study on genetic diversity in many species, including several species of conifer in the world as well as Viet Nam [4-7]. In this study, genetic diversity of natural K. evelyniana populations in Tay Nguyen, Viet Nam was determined. The obtained results will provide the information for conservation, management and restoration of biological diversity of this species in Tay Nguyen in particular and in Viet Nam in general. 2. MATERIALS AND METHODS 2.1. Materials Table 1. Details of K. evelyniana genotypes and populations used in this study. Population code Collection locality Sample number Sample code Latitude ( ◦ N) Longitude ( ◦ E) Elevation (m) Lam Dong Suoi Vang, Lac Duong, Lam Dong 10 Ke1 – Ke10 11° 59’ 58.8" 108° 21’ 59.3” 1464 Hiep An, Duc Trong, Lam Dong 12 Ke11 – Ke22 11° 50’ 14.0” 108° 26’ 35.5” 1390 Da Chais, Lac Duong, Lam Dong 4 Ke23 – Ke26 12°12’04.5” 108°40’06.2” 1485 Dak Lak Hoa Son, Krong Bong, Dak Lak 21 Ke27 – Ke47 12° 25’ 05.2" 108° 22’ 17.1” 1116 Kon Tum Dak Glei, Dak Glei, Kon Tum 23 Ke48 – Ke70 15 0 01’ 17’’ 107o 48’ 04” 1553 Genetic diversity among natural populations of Keteleeria evelyniana Mast. in Tay Nguyen 277 Seventy leaf and bark samples of 70 individuals randomly selected from 219 K. evelyniana trees in Lam Dong, Dak Lak and Kon Tum were used in this study. The fresh samples were kept in a Ziplock bag including silica gel, and then stored at room temperature until use. Information of the samples was showed in Table 1 and Figure 1. Figure 1. Map showing the studying sites of K. evelyniana. The primers in this study were synthesized by the IDT, (Intergrated DNA Technology, USA). The nucleotide sequences of these primers referred from previously studies are listed in Table 2. 2.2. Methods DNA extraction: Total DNA was extracted and purified using the method described by Porebski et al. [8]. The concentration of DNA was determined by a UV-visible light spectrophotometer (UVS 2700, Labomed, USA), and the DNA samples were diluted to 20 ng/µL and used as templates for PCR amplification. PCR_SSR reaction: PCR reactions were performed on the PCR system 9700 (USA) with a total volume of 25 µl. The composition of the reaction and the thermo cycle followed the ones described in Dinh Thi Phong et al. [7]. The SSR fragments were detected on 5 % polyacrylamide gel 1X TAE, then were visualized under UV using BioDocAnalyze (Biometra). KON TUM DAK LAK LAM DONG Dak Glei Hoa Son Da Chais Suoi Vang Hiep An Hoang Sa Islands Truong Sa Islands Phu Quy Island Tho Chu Island Con Dao Island Phu Quoc Island Tran Thi Lieu, Vu Thi Thu Hien, Dinh Thi Phong 278 Table 2. The nucleotides sequences, PCR production sizes and optimum annealing temperatures of SSR markers in this study. No. Primers code Nucleotides Reference Ta ( o C) Size (bp) 1 FRPP91 5‘ GTACTCCCACATAAAATGAGACTT 3’ 3’ CCGAAATACATTGCAGGTTA 5’ [9] 53 100 – 180 2 Cm3 5’ TGGGTTGACCAGTGCTTCT 3’ 3’ ATGCCCAACACCTCATTAGA 5’ [4] 53 120 – 200 3 PeB31BGT 5’ GGCATTGGCTCAACAGA 3’ 3’ TCGTGGAGAGGTACTTCATT 5’ [10] 54 190 – 500 4 Pinus10 5’ CGGGCTGGTATCTCAAGAGT 3’ 3’ ACACACACACACAGAGAGAGAG 5’ [5] 55 285 – 305 5 PRE10 5’ CTGGTCTTGGCCTAAGAATATGAAG 3’ 3’ CATTGGGACGTAAACAACAATACCA 5’ [11] 52 120 – 210 6 PRE13 5’GATGTGTCTTTAGGCTCGTTGC 3’ 3’ AGGGTTAGTAATCACGGCCTGT 5’ [11] 54 170 – 180 7 PRE16 5’ TCCTGCGATGAGTCTCTTTGT 3’ 3’ TCCATTTTTTACTTTTGATAACTTTAC 5’ [11] 52 195 – 490 8 PRE24 5’ GTTTTTTAAATTGGGAAGGCG 3’ 3’ CGTGGGGGAGATAGTGATAGAGT 5’ [11] 54 380 – 400 9 P1 5’ CTCCCTCTATGTGTTTCTCC 3’ 3’ GAAAATCTTTCTACCCTTCCAG 5' [12] 55 300 - 400 10 P5 5’ GTTCGCTAGTTTGTTTGATCCC 3’ 3’ TCCCAGCAAATCCTTGACTC 5’ [12] 53 145 – 160 11 Pt36480 5’TTTTGGCTTACAAAATAAAAGAGG 3’ 3’ AAATTCCTAAAGAAGGAAGAGCA 5’ [13] 52 160 – 160 12 Pt87268 5’ GCCAGGGAAAATCGTAGG 3’ 3’ AGAAGATTAGACATCCAACCC 5’ [14] 56 160 – 160 13 PtTX3026 5’ AATACTTGGGAGGGATAC 3’ 3’AATAGCCAGTTTTGTTTG 5’ [15] 53 130 – 255 14 PtTX3034 5’ TCAAAATGCAAAAGACG 3’ 3’ ATTAGGACTGGGGATGAT 5’ [15] 53 200 – 210 15 RPS1b 5’ GCCCACTATTCAAGATGTCA 3’ 3’ GATGTTAGCAGAAACATGAGG 5’ [16] 54 100 – 100 16 RPS2 5’ CATGGTGTTGGTCATTGTTCCA 3’ 3’ TGGAGGCTATCACGTATGCACC 5’ [16] 54 185 – 210 Data analysis: The parameters including genetic diversity of each population as the average of number of observed alleles (Na), effective alleles (Ne) and private alleles (Ap) per locus, percentage of polymorphic bands (PPB), the Shannon’s genetic diversity index (I) [17], the expected (He) and observed heterozygosity (Ho = number of heterozygous individuals/ total individuals), and the Wright’s inbreeding coefficient (Fis) were analyzed and obtained using the GENALEX 6.3 [18] and FSTAT software [19]. The genetic differences coefficient (Fst) and gene flow (Nm) for each locus were calculated using the formula: Fst = (Ht - Mean He)/ Ht and Nm = [(1/Fst)-1]/ 4, in where He = 1 - ∑(pi), Ht (total expected heterozygosity) = 1 - ∑(tpi)2, (pi is the frequency of the i th allele, tpi is the frequency of the i th allele for the total). Exact tests of Genetic diversity among natural populations of Keteleeria evelyniana Mast. in Tay Nguyen 279 deviation from the Hardy-Weinberg equilibrium for all loci and among populations were performed at the significance level (P) of 0.001. Analysis of molecular variance (ANOVA) was also conducted to calculate level of significant variation among and within populations using the GENALEX 6.3 program. We also constructed a dendrogram base on similarity matrix of 70 K. evelyniana samples using the method of Nei and Li [20] in NTSYS software version 2.0. The bootstrap value was repeated 1000 times and supported by Win-Boot software [21]. 3. RESULTS 3.1. Genetic diversity The sixteen SSR primer pairs were used to assess the genetic diversity for 70 K. evelyniana individuals of three populations in Lam Dong, Dak Lak and Kon Tum. There were 13/16 primer pairs showing polymorphism with an average of polymorphism information content (PIC) and the intra locus gene diversity (Hj) of 0.182 and 0.210, respectively. A total of 39 DNA fragments were amplified with their sizes ranging from 100 bp to 500 bp, of which 35 fragments were polymorphic (accounting for 89.74 %), an average of 2.44 fragments per marker (Table 3). Figure 2 showed the representative results of PCR products from the 70 samples using the Pinus10 marker. Figure 2. The PCR-SSR products of the 70 specimens using Pinus10 on 6 % polyacrylamide gels (numbers 1 –70: the samples from Ke1 to Ke70, M: marker 100 bp). The analyzed parameters such as the average of number of alleles observed (Na), effective alleles (Ne) and private alleles (Ap) per locus, the Shannon’s genetic diversity index (I), the expected (He) and observed heterozygosity (Ho) of each population were showed in Table 4. The results showed that genetic diversity in Lam Dong population (2.063; 1.730; 0.375; 0.558; 0.459 and 0.367, respectively) was the highest and there was in significant difference between Dak Lak and Kon Tum populations. The number of effective alleles per locus, observed and expected heterozygosity in Kon Tum population (Ne = 1.651; Ho = 0.370 and He = 0.346) were higher than those of Dak Lak (Ne = 1.637; Ho = 0.363 and He = 0.333), while the average number of observed alleles and private alleles per locus, Shannon’s genetic diversity index in Dak Lak population (Na = 1.875, Ap = 0.188 and I = 0.495) were higher than those of Kon Tum (Na = 1.750; Ap = 0.063 and I = 0.489, respectively) (Table 4). At the population level, the mean of Shannon’s genetic diversity index of K. evelyniana population was 0.514. This genetic diversity level was higher than that of P. krempfii population (I = 0.377) [6], but lower than that of P. dalatensis population in Tay Nguyen (I = 0.524) [7]. M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 3 9 40 41 42 43 44 45 46 47 4849 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 200 bp 300 bp 400 bp Tran Thi Lieu, Vu Thi Thu Hien, Dinh Thi Phong 280 Table 3. Value PIC, intralocus genetic diversity and the percentage of polymorphic bands of SSR markers. No. Primers Size (bp) PIC Polymorphic bands Monomorphic bands Total bands Percentage of polymorphic bands Intralocus genetic diversity (Hj) 1 FRPP91 100-180 0.113 1 1 2 50 0.240 2 Cm3 120-200 0.482 4 0 4 100 0.359 3 PeB31BGT 190-500 0.174 3 0 3 100 0.118 4 Pinus10 285-305 0.146 2 0 2 100 0.250 5 PRE10 120-210 0.418 5 0 5 100 0.226 6 PRE13 170-180 0.145 2 0 2 100 0.359 7 PRE16 195-490 0.386 4 0 4 100 0.455 8 PRE24 380-400 0.014 2 0 2 100 0.054 9 P1 300- 400 0.237 3 0 3 100 0.234 10 P5 145- 160 0.246 2 0 2 100 0.191 11 Pt36480 160-160 0.000 0 1 1 0 0.000 12 Pt87268 160-160 0.000 0 1 1 0 0.000 13 PtTX3026 130-255 0.118 2 0 2 100 0.345 14 PtTX3034 200- 210 0.146 2 0 2 100 0.250 15 RPS1b 100- 100 0.000 0 1 1 0 0.000 16 RPS2 185- 210 0.277 3 0 3 100 0.278 Sum 100-500 2.902 35 4 39 - 3.359 Mean 0.182 2.188 0.250 2.438 89.74 0.210 Table 4. Genetic diversity of three K. evelyniana populations at 16 SSR markers. Populations Na Ne I Ho He Ap Fis PPB (%) Lam Dong 2.063 1.730 0.558 0.459 0.367 0.375 - 0.234 81.25 Dak Lak 1.875 1.637 0.495 0.363 0.333 0.188 - 0.065 81.25 Kon Tum 1.750 1.651 0.489 0.370 0.346 0.063 - 0.047 75.00 Mean 1.896 1.673 0.514 0.397 0.349 0.208 - 0.115 79.17 At the species level 2.438 2.027 0.677 0.401 0.418 - - 81.25 Notes: Na: the average number of alleles per locus; Ne: number of effective alleles per locus; I: Shannon’s genetic diversity index; Ho and He: the observed and expected heterozygosity; Ap: number of private alleles per locus; Fis: Wright’s inbreeding coefficient with p < 0.05; PPB%: percentage of polymorphic bands. The results in Table 4 also showed that among the private alleles (Ap) found in all 3 populations, the highest number was in Lam Dong population (0.375) and the lowest was in Kon Tum population (0.063). The fixation index values of all studied populations were negative (Fis < 0) with ranging from - 0.047 of Kon Tum population to - 0.234 of Lam Dong population. The observed heterozygosity of K. evelyniana populations were higher than the expected heterozygosity (Ho > He). This result indicated that the excess heterozygosity of K. evelyniana species in Tay Nguyen could be attributed to the phenomenon of cross-pollinating between populations. At the species level, genetic diversity of K. evelyniana was expressed by their effective allele number per locus Ne = 2.027, Shannon’s genetic diversity index I = 0.677 and expected heterozygosity He = 0.418 (Table 4). Comparison to some other conifer species in Tay Nguyen showed that the expected (He) and observed heterozygosity (Ho) values of K. evelyniana (0.418 Genetic diversity among natural populations of Keteleeria evelyniana Mast. in Tay Nguyen 281 and 0.401) were the higher than those of P. krempfii (0.234 and 0.310) [6]. It’s expected heterozygosity value found the lower than that of P. dalatensis (He = 0.418 vs 0.524), however it’s observed heterozygosity value was the higher (Ho = 0.401 vs 0.234) [7]. In order to further evaluate genetic diversity degree of K. evelyniana, genetic parameters for each SSR marker are also analyzed. The analysis indicated that the average of observed (Ho) and expected heterozygosity (He) values of K. evelyniana population were 0.423 and 0.324, respectively (Table 5). Comparison of heterozygosity level of species in the genus Keteleeria showed that K. evelyniana species in Tay Nguyen (Ho = 0.423, He = 0.324) were lower than those of K. davidiana var. formosana species in Taiwan (Ho = 0.68, He = 0.82) and (Ho = 0.63, He = 0.78) in study of Ho et al. [22]. The results in Table 5 also showed that the average of gene flow (Nm) of K. evelyniana was 5.423. The marker PtTX3026 gave the highest Nm (Nm = 34.882 and Fst = 0.007) and the marker Cm3 was the lowest (Nm = 0.312 and Fst = 0.445). Compared with P. krempfii species in Tay Nguyen, gene flow level of K. evelyniana species was the higher (2.315 vs 5.423, respectively) [6]. Table 5. The genetic parameters of all K.evelyniana populations for 16 SSR markers. Primers Na Ne I Ho He Fis Fst Nm FRPP91 2.00 1.688 0.564 0.647 0.384 -0.686 0.123 1.776 Cm3 1.80 1.669 0.514 0.134 0.361 0.629 0.445 0.312 PeB31BGT 2.20 1.769 0.625 0.505 0.405 -0.249 0.104 2.153 Pinus10 2.00 1.973 0.686 0.819 0.493 -0.661 0.008 30.856 PRE10 2.20 2.132 0.761 0.519 0.523 0.007 0.235 0.814 PRE13 2.00 1.847 0.647 0.695 0.455 -0.527 0.030 8.096 PRE16 1.80 1.333 0.365 0.214 0.229 -0.433 0.376 0.416 PRE24 2.00 2.000 0.693 1.000 0.500 0.064 0.065 3.620 P1 1.60 1.346 0.318 0.198 0.211 -1.000 0.138 1.563 P5 1.00 1.000 0.000 0.000 0.000 0.062 0.158 1.337 Pt36480 1.00 1.000 0.000 0.000 0.000 - - - Pt87268 2.00 1.981 0.688 0.763 0.495 - - - PtTX3026 2.00 1.566 0.499 0.313 0.330 -0.542 0.007 34.882 PtTX3034 2.00 1.872 0.654 0.662 0.462 0.052 0.322 0.526 RPS1b 1.80 1.579 0.486 0.302 0.334 - - - RPS2 1.00 1.000 0.000 0.000 0.000 0.095 0.377 0.412 Mean 1.775 1.610 0.469 0.423 0.324 -0.245 0.184 5.423 Notes: Na: average of number of alleles per locus; Ne: number of effective alleles per locus; I: Shannon diversity index; Ho and He: the observed and expected heterozygosity; Fis: Wright’s inbreeding coefficient with p < 0.05; Fst: coefficient of genetic differences; Nm: gene flow. 3.2. Population structure Molecular variance (ANOVA) analysis among K. evelyniana populations using SSR markers indicated that 65.35 % of the total genetic diversity was distributed within groups and only 34.65 % was attributed to differences between regions (with p value < 0.001) (Table 6). The low variability between populations was also reported by Tam et al., 2013, in which the genetic variation was found in the populations of Glyptostrobus pensilis of Viet Nam [23]. Tran Thi Lieu, Vu Thi Thu Hien, Dinh Thi Phong 282 Table 6. Analysis of molecular variance among/within K. evelyniana populations. Source of variance Degree of freedom Sum of squares Variance components Total variation (% ) p value Among population 2 65.259 1.298 34.65 < 0.001 Within population 67 164.112 2.449 65.35 Genetic differences between populations of K. evelyniana were calculated based on alleles frequency comparison of markers between pairwise populations and showed in Table 7. The average genetic difference level between studied populations was low with Fst = 0.118. The lowest one (Fst = 0.092) was between Lam Dong and Dak Lak population and the highest (Fst = 0.137) was between Dak Lak and Kon Tum populations (Table 7). When comparison with some other coniferous species, this value of K. evelyniana in Tay Nguyen (Fst = 0.118) was found to be lower than P. dalatensis (Fst = 0.287) [7], P. resinosa (Fst = 0.280) and P. radiata (Fst = 0.14) [10, 24], and higher than P. cembra (Fst = 0.02) [25]. Table 7. Value of genetic differences between pairwise of populations K. evelyniana. Lam Dong Dak Lak Kon Tum Lam Dong Dak Lak 0.092 Kon Tum 0.105 0.137 The low in both molecular variance (34.65 %) and genetic difference (Fst = 0.118) between the populations of K. evelyniana in Tay Nguyen indicated the studied species is relatively conservative in its genome and only slight variation can be occurred depending on geographical features. 3.3. Genetic relationships among 70 K. evelyniana samples A UPGMA dendrogram constructed based on similarity matrix with SSR markers (Figure 3) divided 70 K. evelyniana samples into two main groups (I and II) with their genetic similarity coefficient ranged from 76.5 % (Ke26 and Ke44) to 99 % (Ke23 and Ke25). Each main group also divided into two subgroups. Subgroup I.1 included 23 samples collected in Kon Tum (Ke48 – Ke70) with their genetic similarity coefficient ranged from 84 % to 98.6 %. The second subgroup I.2 included 21 samples originated from Dak Lak (Ke27 – Ke47) with their genetic similarity coefficient ranged from 81.4 % to 98.3 %. The subgroup II.1 and II.2 included 12 samples of Hiep An (Lam Dong) (Ke11 – Ke22) and 14 samples of Suoi Vang and Da Chais (Lam Dong), respectivelly. The obtained results obviously showed that the samples in the same geographic regions were clustered into the some subgroups. The genetic similarities between samples in the dendrogram (from 76.5 % to 99 %) were also consistent with population structure analysis above (genetic variation among populations 34.65 % and genetic difference Fst = 0.118). Genetic diversity among natural populations of Keteleeria evelyniana Mast. in Tay Nguyen 283 Figure 3. UPGMA dendrogram based on similarity genetic coefficient of 70 K. evelyniana samples analysis SSR markers with the bootstrap values repeat 1000 times (Note: a: samples in Kon Tum; b: samples in Dak Lak; c: samples in Hiep An (Lam Dong); d: samples in Da Chais and Suoi Vang (Lam Dong). 4. CONCLUSION In this study, genetic diversity indicators of K. evelyniana within and between popullations were analysed using SSR markers. They were the highest in Lam Dong (Na = 2.063; Ne = 1.730; Ap = 0.375; I = 0.558; Ho = 0.459 and He = 0.367) and not significant different between Dak Lak and Kon Tum populations. The rate of cross-pollination among the three populations was high (Fis < 0), indicating a very low rate of inbreeding in each population occurred. The number of private alleles (Ap) were found in all Lam Dong (0.375), Dak Lak (0.188) and Kon Tum (0.063) populations using SSR markers. The gene flow (Nm) also occurred in the populations of K. evelyniana with Nm = 5.423. The total level of molecular changes between populations was relatively low (4.65 %) and high within populations (65.35 %). The genetic similarity coefficient of K. evelyniana in Tay Nguyen ranged from 76.5 to 99 %. Based on Fis value, gene flow rate and total level of molecular variance analysis, it can conclude that the genetic diversity of K. evelynianais in Tay Nguyen is alarming, and conservation and management strategies for this species are urgently needed. Acknowledgements: This research was financially supported by the Tay Nguyen 3 Program for the Project number TN3/T15. 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